Membrane trafficking, viral infections and development involve membrane fusion. While some viral fusogens are well characterized, most fusogens remain unknown. Recently we explored the post-mitotic nuclear envelope reassembly using cell-free system. Liposomes pre-incubated with frog egg cytosolic extract were found to bind to chromatin and fuse at its surface in a GTP-dependent manner suggesting that vesicle targeting to the chromatin and fusion initiation in nuclear assembly do not require transmembrane proteins. We also explored fusogens that mediate cell-cell fusion in C. elegans development. While most of these fusion events involve fusogen EFF-1, we identified an additional and structurally similar fusogen AFF-1 that is responsible for uterine cells fusion to the gonadal anchor cell that establishes a uterine-vulval tube. AFF-1 is required and sufficient for this fusion in vivo and, when expressed in exogenous cells, fuses them. EFF-1 and AFF-1 are the founding members of a family of nematode fusogens. Diverse fusions converge to pathway that includes the conserved early intermediate - hemifusion stalk. Our new analysis suggests that stalk formation involves the point-like protrusions. This pre-stalk intermediate has energy lower than that of the stalk and, therefore, does not limit the fusion. The point-like protrusion completes the fusion-through- hemifusion model of membrane merger.[unreadable] [unreadable] Cell fusion is fundamental for reproduction and organogenesis. Most of cell fusion events in C. elegans are mediated by the EFF-1 fusogen that we explored in our earlier work. However, fusion between the anchor cell and the utse syncytium that establishes a continuous uterine-vulval tube, a passage for eggs to exit the vulva, proceeds normally in eff-1 mutants. By isolating mutants where the anchor-cell fails to fuse, we identified aff-1. AFF-1 ectopic expression results in fusion of cells that normally do not fuse in C. elegans. The necessity of AFF-1 for specific fusion events, combined with its ability to promote ectopic cell fusions, suggests that this protein acts directly in the cell fusion process. To explore this further, we expressed the AFF-1 protein in heterologous cultures of Sf9 insect cells and found this protein to be even more potent fusogen than EFF-1 at similar surface densities. The ability of AFF-1 to fuse heterologous cells confirmed that this protein is an actual fusogen rather than a regulator of fusion reaction. AFF-1 and EFF-1 differ in their expression patterns but demonstrate a striking conservation in the position and number of all 16 cysteines in their ectodomains. While there are clear homologs in other nematodes, AFF-1 and EFF-1 exhibit only minor similarity to proteins from other vertebrates and invertebrates. In summary, EFF-1 and AFF-1 are the founding members of a family of fusogens in C. elegans and probably in other nematodes. Our findings indicate that different cell-cell fusion events in C. elegans development utilize distinct fusogens and that EFF-1 expressing cells do not fuse with AFF-1 expressing cells. The regulated expression of distinct fusogens might establish the formation of developmental barriers between adjacent syncytia that may represent a general characteristic of developmental fusion.[unreadable] [unreadable] Membrane fusion stage of post-mitotic re-assembly of nuclear envelope is very different from cell-cell fusion. Protein fusogens that initiate membrane fusion at the early stage of tightly controlled nuclear assembly are yet to be identified. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on the nuclear reconstitution in cell-free system. Mixing of soluble interphase cytosolic extract and membrane vesicles from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced membrane vesicles and cytosol with protein-free phosphatidylcholine liposomes (LS) that were pre-incubated with interphase cytosol. While later stages of nuclear assembly yielding functional nucleus did not proceed without integral proteins of membrane vesicles, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus, in contrast to post-fusion stages, fusion initiation in NE assembly, like membrane remodeling in budding and fission, does not require transmembrane proteins.[unreadable] [unreadable] Diverse fusions appear to converge to membrane remodeling pathway that includes the conserved early intermediate hemifusion stalk. The structure and energies of lipid intermediates preceding the fusion stalk formation have remained unexplored. If these pre-stalk intermediates have higher energy cost than the stalk, their formation might limit the rate of the whole fusion reaction. To form a stalk, the membranes have to establish, at least locally, a dehydrated contact allowing them to perturb the continuity of their surfaces and merge, without exposure of the hydrophobic moieties of lipids to the aqueous surrounding. Such membrane contact requires overcoming the resistance of the powerful short range repulsion forces. Due to these hydration forces the conventional wave-like membrane bulges approaching each other would have energies of hundreds of kT and thus are very unlikely. To overcome this problem we suggest an energetically feasible structure of a pre-stalk intermediate that has a sharp tip allowing for establishment of a point-like rather than extended dehydrated contact between the membranes. We analyzed the overall energy of the PLP using the elastic tilt-splay model for the membrane deformations and the hydration force model for the inter-membrane repulsion and found that interplay between the splay of the lipid hydrocarbon chains and their tilt with respect to the monolayer surface decreases elastic energy costs of PLP to modest values. Further, we obtained that due to the shape of PLP, along with the discreteness of the hydration centers, the hydration repulsion energy of PLP formation also remains in the range of few tens of kT. For the relevant lipid compositions of membrane monolayers, the overall energy of PLP is lower than that of the fusion stalk. Consequently, PLP does not limit the rate of hemifusion and the lipid dependency of hemifusion is determined by the stalk and the hemifusion diaphragm. Moreover, a point-like dehydrated contact between PLP and the target membrane facilitates stalk formation. We consider the new pre-stalk intermediate to be an important addition to the fusion-through-hemifusion pathway.